Academic Unit of Clinical and Experimental Sciences and the NIHR Southampton Respiratory Biomedical Research Unit, Southampton University Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton University Hospital, Southampton, United Kingdom.
PLoS One. 2013 Sep 11;8(9):e72502. doi: 10.1371/journal.pone.0072502. eCollection 2013.
Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.
支气管上皮的慢性损伤和修复是哮喘的特征。我们之前曾报道，表皮生长因子（EGF）——上皮修复的关键因子——体外刺激正常的支气管上皮细胞可增强中性粒细胞聚集的机制，从而促进急性损伤期间的中性粒细胞防御，但可能会增强慢性气道疾病中的炎症。我们现在试图：（i）确定在 EGF 和其上皮受体过度表达的哮喘中，这种依赖于 EGF 的促中性粒细胞活性是否增加；（ii）阐明这种哮喘上皮-中性粒细胞相互作用的一些机制。从健康受试者、轻度哮喘患者和中重度哮喘患者（Mod/Sev）中分离原代支气管上皮细胞（PBEC），用 EGF 刺激，模拟修复上皮。评估条件培养基（EGF-CM）中的中性粒细胞趋化和抗凋亡活性以及炎症介质的产生。EGF 诱导上皮产生具有中性粒细胞趋化（p<0.001）和促生存（p=0.021）活性的可溶性介质，这与哮喘的临床严重程度相关（趋势 p=0.010 和 p=0.009）。这与增强的 IL-6、IL-8、GM-CSF 和 TNF-α释放相关，并且使用 Mod/Sev 哮喘患者的 EGF-CM 进行的细胞因子中和实验表明 GM-CSF 在中性粒细胞存活中起作用（p<0.001）。用骨髓限制的 I 类磷脂酰肌醇 3-OH 激酶（PI(3)K）同工型的特异性抑制剂预处理中性粒细胞表明，Mod/Sev 哮喘患者的 EGF-CM 依赖于 γ（p<0.021）但不依赖于 δ 同工型，而中性粒细胞存活需要多种 I 类 PI(3)Ks。EGF 诱导的趋化活性，但不是促生存活性，涉及中性粒细胞中的 RhoA 信号转导（p=0.012）。在哮喘中活性上调的 EGF 诱导来自哮喘患者的体外上皮产生促中性粒细胞活性；这些与哮喘严重程度相关，在中重度哮喘中，涉及 I 类 PI(3)Kγ 信号转导，为中性粒细胞型哮喘提供了一个潜在的治疗靶点。
